Generation of Mature Toxoplasma gondii Bradyzoites in Human Immortalized Myogenic KD3 Cells

Toxoplasma gondii is a zoonotic protozoan parasite and one of the most successful foodborne pathogens. Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue. Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models. In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited. We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites. Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo–like features, including pepsin resistance, oral infectivity, and antifolate resistance. In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics. In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO2 levels. We also discuss critical aspects of the procedure and suggest improvements. Key features • This protocol describes a scalable human myotube-based in vitro system capable of generating encysted bradyzoites featuring in vivo hallmarks. • Bradyzoite differentiation is facilitated through CO2 depletion but without additional artificial stress factors like alkaline pH. • Functional maturation occurs over four weeks.

This protocol is used in: Nat.Commun.(2022), DOI: 10.1038/s41467-022-28730-w Toxoplasma gondii is a zoonotic protozoan parasite and one of the most successful foodborne pathogens.Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue.Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models.In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited.We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites.Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo-like features, including pepsin resistance, oral infectivity, and antifolate resistance.In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics.In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO2 levels.We also discuss critical aspects of the procedure and suggest improvements.

Rat tail collagen
Collagen I from rat tail dissolved at a stock concentration of 0.1 mg/mL in 100 mM acetic acid.Use 1:6.67 in water.Store at 4 °C.

Procedure
General description of the procedure and notes Generating mature T. gondii cysts in KD3 human myotubes involves three steps (Figure 1): (i) set up a KD3 myoblast culture, (ii) differentiation of these cells into KD3 myotubes, and (iii) infection of myotubes with tachyzoites and the differentiation of parasites to bradyzoites.We also suggest experiments to confirm the maturation of cysts.

(i) Myoblast culture
Handling of KD3 myoblasts is adapted from the procedure described by Shiomi et al. (2011).KD3 myoblasts appear similar to endothelial cells (Figure 2A) and are slightly heat-sensitive (avoid prolonged temperatures higher than 37 °C during cultivation).During maintenance of the myoblasts, it is imperative to keep the main culture subconfluent (<70%), as cell-to-cell contact appears to act as a trigger for differentiation into myotubes.Before the monolayer reaches this density, the cells are subcultured (split).The medium is exchanged for warm phosphatebuffered saline without magnesium and calcium.Pre-warmed trypsin-EDTA solution is added to cover the monolayer and the culture is incubated at 36.7 °C until cells detach.The trypsinization reaction is stopped by adding myoblast growth medium, and a homogenous cell suspension is generated by pipetting.At this point, the cell density can be determined by counting.The suspension is distributed to suitable cell culture vessels, which can optionally be coated with collagen to enhance myotube attachment.Do not aim for a confluency below 5%, as this suppresses myoblast growth.Incubate cultures at 36.7 °C at 5%-10% CO2.
Since cell-to-cell contact cannot be prevented even at low confluency subculturing, KD3 myoblasts may unintentionally start to differentiate to myotubes, and the culture may exhibit a mixture of both cell types (Figure 2C).We often observe this with increased confluency; the growth rate drastically declines, and the culture becomes unsuitable for further use.The number of usable passages of the culture thus highly depends on handling, and we recommend monitoring cell behavior in correlation with the respective passage number.Generally, the number of passages should not exceed 16 of a freshly thawed vial.An occasional selection for the presence of the transgenes might be advisable.The cells are resistant to G418 (400 µg/mL) and puromycin (0.5 µg/mL) (Shiomi et al., 2011).
Long-term KD3 myoblast storage is done in liquid nitrogen in freezing medium.Use a controlled freezing rate apparatus or an isopropanol chamber to ensure a slow and steady temperature drop.For thawing, quickly thaw in a 37 °C water bath, replace the freezing medium with growth medium, and distribute cells to a cell culture vessel.Incubate at 36.7 °C at 5%-10% CO2 until the culture is ready for subcultivation.We usually use 0.3 mL of medium per cm 2 cell culture vessel.(ii) Myoblast differentiation KD3 myoblasts are progenitor cells that undergo myogenesis when induced by serum starvation (Shiomi et al., 2011).Growth medium is replaced with myoblast differentiation medium at a confluency of 20%-50% (Figure 2A).Cells fuse and form multinucleated tubes over the course of five days.Often, myoblasts replicate once or twice before they fuse (Figure 2B).The differentiation efficiency depends on the general potency and passage number of the myoblast culture.

(iii) Tachyzoite infection and bradyzoite maturation
In general, every cystogenic strain of T. gondii can be used for infection, and a selection of type I, type II, and type III strains were tested directly for their ability to generate DBA-positive and SAG1-negative cysts (Christiansen et al., 2022).For a fully matured, in vivo-like cyst culture, we recommend the use of type II or type III strains.We frequently cultivate a Pru-∆hxgprt tdTomato (Chtanova et al., 2008) strain.Furthermore, we recommend using a fluorescent protein-expressing strain to easily monitor the cyst development, since the cysts may be difficult to find using brightfield or phase-contrast microscopy.It is also highly beneficial to use a tachyzoite strain that has been recently re-differentiated from either a cyst-containing tissue homogenate (Dubey, 1998) or a previous in vitro cyst culture, as long-term tachyzoite cultures lead to low differentiation efficiencies (Colos-Arango et al., 2023).

Published: Jan 05, 2024
The myotube culture is infected with 7.2 × 10 3 tachyzoites per cm 2 surface area in cyst medium.The culture is incubated at 36.7 °C and ambient CO2 concentration.Ambient CO2 levels limit the rate of parasite pyrimidine biosynthesis and facilitate stage conversion (Bohne and Roos, 1997).After infection, the culture needs to be monitored daily and the medium is replaced every second to third day.Parasite stage-conversion depends on the quality of the myotubes and the cystogenicity of the strain and will start immediately post-infection.However, longterm tachyzoite cultures with a decreased cystogenic potential may require additional washing steps if parasite egress is apparent to prevent overgrowth of the culture with tachyzoites.After four weeks, the cysts are largely functionally mature (Christiansen et al., 2022).Throughout the experiment, myotubes move and change their shape, which can lead to cell detachment.Furthermore, throughout the culture period, cysts grow in size, new cysts will emerge from egressed bradyzoites, and cysts may also dissipate.

Bench protocol
All parasite and muscle cell handling should be done in a sterile laminar flow BSL-2 workbench.

Application of the protocol
The method can be helpful in many applications, considering the different characteristics to be studied and the scale required for their analysis.While the cyst wall starts to develop within the first week of differentiation as seen by DBA staining (Christiansen et al., 2022), antifolate tolerance fully developed only after four weeks.Further, resistance to pepsin digestion is increasing throughout four weeks of maturation.Myotubes are a natural host cell type for T. gondii and thus may provide a more relevant system to investigate parasite interactions with the host cell than, for instance, HFF.Downstream applications require suitable cyst isolation protocols.We performed an LC-MS-driven metabolic characterization of in vitro bradyzoites.To this end, we isolated tissue cysts using DBA-and streptavidin-coated magnetic beads from syringe-passaged infected KD3 myotubes (Christiansen et al., 2022).This isolation procedure can be performed at 4 °C and does not require detergents, polymers, or other MS-incompatible chemicals.
For other applications such as imaging, intact cysts can also be isolated using a percoll gradient (Watts et al., 2017).Single bradyzoites have been obtained using either pepsin (Dubey, 1998)  In the last 10 years, more than 135 studies in PubMed have mentioned the use of mice in the context of T. gondii bradyzoites.Assuming an average of 100 mice per study and an unknown number of mice that are routinely infected to have a constant supply of tissue cysts, this adds up to several thousand animals.A significant number of those could have been replaced if adequate in vitro systems like this one would have been applied.We hope that this protocol encourages the community to consider its usefulness instead of mouse experiments, where appropriate, thereby contributing to the 3R principle.

Figure 1 .
Figure 1.Overview of the protocol steps.(A) Timing of the cultivation of in vitro T. gondii cysts.KD3 myoblasts are seeded into desired cell culture vessels.When they reach a maximum confluency of 50%, differentiation is induced.After five days, the cells feature longer tube-like multinucleated cells, which are hallmarks of the myotube stage.Myotubes are infected with cystogenic T. gondii parasites.Over the course of four weeks of CO2 depletion, tachyzoites differentiate into bradyzoites and mature to in vitro cysts.(B) Representative pictures of the same culture at relevant developmental stages.Prugniaud parasites express the red fluorescent protein tdTomato.Scale bar indicates 100 µm.

Published: Jan 05, 2024 Figure 2 .
Figure 2. KD3 culture at various stages.(A) Healthy KD3 myoblast culture at the right confluency for myotube induction.Scale bar indicates 500 µm.(B) KD3 myoblast cultures at the maximum density allowed before splitting.(C) Improper handling or high passage number of the KD3 culture creates a mixture of myoblasts and emerging myotubes.Scale bar indicates 100 µm.(D) KD3 myotubes five days post induction.Scale bar indicates 100 µm.
(Christiansen et al., 2022)mi et al., 2018;Spalenka et al., 2018;Han et al., 2020)be directly applied to the monolayer and may provide a means of removing immature bradyzoites before purification.Given the need for new drugs and drug targets, research efforts in the field of drug discovery have intensified over the last years(Dittmar et al., 2016;Adeyemi et al., 2018;Spalenka et al., 2018;Han et al., 2020).However, none of these studies integrated matured cysts into the screening process.Our method can be adapted into a 96-well plate format suitable for drug screens.Compounds that are cidal to bradyzoites would prevent regrowth of redifferentiated tachyzoites, which are easily detectable by fluorescence or plaque assay.In vitro bradyzoites develop tolerance towards antifolates and other antiparasitics in a time-dependent manner(Christiansen et al., 2022).